Outline

Background: Gene therapy may be a valuable tool for acute myeloid leukemia (AML) to treat patients refractory to conventional chemotherapy. However, there are no vectors available that sufficiently and specifically transduce this cell type. Vectors can be targeted to cell type specific receptors. We have developed a screening system based on random peptide libraries displayed on adeno-associated virus type 2 (AAV) (MÃ¼ller et al., Nat. Biotechnology, 2003). Such libraries consist of up to 108 different viruses that display a peptide with random sequence within a capsid region that mediates binding of the virus particle to cellular receptors. Targeted AAV vectors that specifically transduce the cell types of interest can be selected from such libraries. Here we report the selection of a random AAV display peptide library on AML cell lines (Kasumi-1, HL 60).

Methods: Oligonucleotide inserts encoding a random seven amino acid peptide were cloned into the AAV-library plasmid followed by the production of AAV library transfer shuttle AAV in 293T cells. To produce the final library, 293T cells were infected with shuttle AAV at an MOI of 1 to ensure encoding of displayed peptides by the packaged DNA. The target AML cells were then infected with the AAV library. Internalized particles were amplified by superfinfection with adenovirus type 5. Amplified clones were recovered and subjected to 2 more rounds of selection. The peptide inserts of the enriched capsid mutants were analyzed indirectly by DNA sequencing.

Results: After 3 rounds of selection, enrichment of a peptide motif within the selected AAV capsids was observed on both AML cell lines. The selected virus capsids were used for production of recombinant AAV vectors harboring a GFP reporter gene. Such targeted vectors transduced the target AML cells they have been selected on to almost 100%, which is up to 60x more efficient compared to a random clone of the initial, unselected library. One of the selected clones also efficiently transduced all members of a panel of AML cell lines other than the cell line used for selection. In contrast, only a small fraction of a panel of non-hematopoeietic cell lines was transduced by the selected clone, suggesting lineage specificity of the clone’s transduction behaviour. Binding of this clone to leukemia cells seemed to be independent of the natural AAV-2 receptor as it could not be blocked by soluble heparin and transduction could also be shown in heparan sulfate proteoglycan-negative pgsD677 cells. Targeted transduction of leukemia cells could be blocked with empty capsids displaying the targeting peptide but not with wild type capsids suggesting that the targeted AAV use both attachment and internalization pathways that are distinct from the pathways used by wild type AAV.

Conclusion: We conclude that the AAV mutant selected here may be used as a valuable tool to target therapeutic genes to acute myeloid leukemia cells.